Soluble extracts of neonatal rat skeletal muscle contain polypeptides which enhance the neurite-outgrowth and stimulate cholinergic development in dissociated spinal neurons in culture. Acetylcholine synthesis enhancing activity is found in four chromatographically distinct species with apparent molecular weights of 55,000, 17,000, 7000 & 1500 daltons. These factors represent the major cholinergic neurotrophic factors in skeletal muscle extract which enhance in vitro cholinergic development. Most of the acetylcholine synthesis enhancing activity is associated with the 17,000 and 1500-dalton species although the addition of the 55,000-dalton factor to saturating concentrations of these factors produces an additive response. Partial purification of the 55,000, 17,000-dalton cholinergic species has shown that these factors are basic glycosylated polypeptides, while partial purification of the 1500-dalton component has shown that this factor is an acid stable peptide. Such factors may also be important to the normal maintenance and development of motor neurons in vivo and deficiencies in these factors or impairment of their action may lead to motor neuron disease. As a first step in the validation of the role of these factors in vivo, our present efforts are directed at purificaiton of the 17,000-dalton component, using conventional and high resolution biochemical techniques. Preparation of antiantibodies to the purified factor and characterizing the in vitro action of the purified factor on ventral spinal cord cultures.